SUMMARY To prevent deadly CoV infections we propose to study the molecular basis of coronavirus- induced lung edema and its resolution, through the identification of (i) Signaling pathways resulting in severe lung disease to inform inhibitors as antiviral candidates; and (ii) Virus virulence genes. Deletion of these genes will lead to attenuated vaccine candidates. Three aims are proposed: Aim 1. To determine the factors involved in edema induction and resolution during CoV infection. We have shown that both E and 3a proteins of SARS-CoV, and proteins E and 5 of MERS-CoV include two sequence domains involved in virulence, one containing a PDZ binding motif (PBM), and another one encoding ion channel (IC) activity. The binding of the SARS-CoV E protein PBM to proteins containing the PDZ motif causes Acute Respiratory Disease Syndrome in infected animals. The importance of the PBM is likely associated with its ability to bind to more than 400 cellular proteins and, therefore, to regulate many cell signaling pathways. We will study the whole-proteome interactions between PBMs in MERS-CoV, and cellular PDZs. This interactome will be the basis for the identification of peptides interfering with PBM-PDZ binding, using peptide libraries. The mechanism of inflammasome activation by MERS-CoV proteins with IC activity will be studied. Edema resolution is possible by two enzymatic activities: epithelial sodium channel activity (ENa+C) and Na+/K+ ATPase that move Na+ ions from the alveolar fluid into the interstitium promoting water elimination. We showed that SARS-CoV E protein binds Na+/K+ ATPase and have postulated that this binding reduces Na+/K+ ATPase activity, leading to lung edema; this will be investigated in this project. Aim 2. We propose to identify viral and host non-coding RNAs involved in MERS-CoV pathogenesis and lung inflammation, as potential targets in antiviral and anti-edema strategies. Aim 3. To develop safe live-attenuated vaccines for MERS-CoV. The construction of MERS-CoVs defective in propagation, and the generation of attenuated, dissemination competent rMERS- CoVs are proposed. Maximizing biosafety and genetic stability of the vaccine candidates are main goals of the project.